Infants and children <2 yrs are the age group with the highest rates of iron deficiency anemia. Provision of sufficient dietary iron to this age group is a challenge, and in-home iron fortification of complementary foods using micronutrient powders is a promising approach. However, WHO has recommended against untargeted use of in-home micronutrient powders that contain the iron RDA for a child in a single dose in malarial areas, until their safety is proven. Research priorities from the U.S. NIH Technical Working Group (2009) "Considerations for the safe and effective use of iron interventions in areas of malaria burden" include: i) effect of iron delivered through different means and formulations on gut microflora;ii) extent to which iron in the gut lumen favors growth of enteric microorganisms with a pathogenic potential. Therefore, we plan to investigate the effect of iron provided in in-home fortification on the infant gut microbiome, using complementary in vitro and in vivo lines of investigation. The focus is on Salmonella spp., as the pathogen most often implicated in studies of bacteremia in severe malaria. The study aims are: i) in vivo, to determine if in-home fortification using an iron-containing micronutrient powder in Kenyan infants at high risk for diarrheal disease and malaria will modify the composition and metabolic activity of the gut microbiota to favor growth of bacterial enteropathogens, and identify the specific enteropathogens that emerge;ii) in vitro, using immobilized infant fecal microflora in a continuous colonic fermentation model inoculated with the specific pathogens identified in the in vivo studies, as well as a cell culture Salmonella infection model, to determine the mechanisms by which colonic iron selects for bacterial enteropathogens, including iron dose, iron form and the molecular and metabolic response of the gut microbiota to iron. To thoroughly characterize the global impact of iron fortification on the gut microbiota and specifically, on enteropathogens, we will apply TTGE, POR, high-throughput 16S rRNA gene sequence analysis, SCFA production by HPLC as well as metagenomic approaches to measure genotype effects. PUBLIC HEALTH RELEVANCE: This project should provide basic knowledge on the interactions between dietary iron and the infant gut microbiota in order to design safe and effective in-home fortification strategies to control iron deficiency anemia in infants in tropical countries with high burdens of infectious disease.